1. Field of the Invention
The invention is directed to a method for the configuration a laser scanning microscope for a raster image correlation spectroscopy measurement and to a method for carrying out a raster image correlation spectroscopy measurement and for evaluating sampling values of a raster image correlation spectroscopy measurement. Further, the invention is directed to a correspondingly arranged control unit, a correspondingly arranged laser scanning microscope, and a correspondingly arranged computer program.
2. Description of Related Art
Fluorescence correlation spectroscopy (FCS) can be used to examine variable material concentrations in the microscopic size range which are brought about by diffusion processes and other transport processes in a sample. This makes it possible to observe physical and biological transport processes in an individual volume, or through an individual volume, with a diameter of about 200 nm. In order to make assertions about the transport processes in the sample, correlations with the fluorescence measurement data are determined and mathematical transport models are fitted to these correlations, for example, by means of curve fitting. In this way, diffusion constants, for example, can be determined from the adapted models. The transport models are typically mathematical functions whose parameters are adapted.
A spatial resolution of microscopic transport processes is achieved by means of scanning fluorescence correlation spectroscopy (S-FCS), also known as image correlation spectroscopy (ICS). In this way, temporal orders of magnitude from seconds to minutes can be tracked. Tracking which is spatially resolved in two or three dimensions within a cell or between membranes separated by cells within a time range from microseconds to milliseconds is made possible by raster image correlation spectroscopy (RICS). In this case, the sample is optically raster-scanned in two or three dimensions. A laser scanning microscope is advisably used for scanning correlation spectroscopy.
During the optical scanning movement in a RICS measurement, digital scan values (sampling values, samples) are acquired electronically along a first scan direction (scan line) at a typically constant sampling value recording frequency (sampling frequency). Every pixel value is determined from one or more sampling values. An interval within which a quantity of sampling values is associated with a determined pixel, or the period or frequency of such an interval, is designated as the pixel time. The scanning along the first scan direction is carried out repeatedly after moving the scanning beam along a second scan direction (scan column) so that a sequence of pixel lines is acquired. Intervals or periods of successive pixel lines are referred to as the line time. In case of a temporally nonlinear scanning movement along the first scan direction and a constant sampling frequency, the time increments are varied in the form of the pixel time for distortionless acquisition with constant spatial increments.
Errors in fitting the mathematical transport models to the correlations which are determined on the basis of the sampling values or pixel values depend directly on the suitability of the sampling values or pixel values derived therefrom for an evaluation of this kind. The suitability of the sampling values or pixel values in turn depends in a complicated manner on the size of the sample region to be examined, the density of the fluorescence markers in the sample, impurities in the sample medium with fluorescence markers, the scan frequencies and scan speeds, the optical properties of the microscope, the type of scanning movement, the illumination intensity, and the sensitivity of the detectors. The expression ‘suitability’ comprehends statistical and systematic error sources.